Ribonucleotide reductase (RR, EC 1.17.4.1), which reduces ribonucleotides to 2'-deoxyribonucleotides, is essential to de novo DNA synthesis and plays a direct role in regulating DNA replication (Cory, 1988, Adv. Enzyme Regul. 27:437-455). Class I, the most common form of RR, comprises two different subunits, a larger subunit denoted R1 and a smaller subunit, denoted R2. The active enzyme is a dimer of dimers, denoted R1.sub.2 R2.sub.2. Mouse R1 (mR1) and mouse R2 (mR2) have molecular masses 90 kDa and 45 kDa, respectively. Class I RRs catalyze the reduction of nucleoside diphosphates. The NDP substrate binding site, as well as two kinds of allosteric sites, are located on the R1 subunit. R2 contains two high spin Fe (III), as well as 0.5-1.0 stable tyrosine radical per subunit. The catalytic reaction is believed to involve long-range electron transfer between the tyrosine radical and the substrate site. More complete descriptions of this enzyme may be found in three recent reviews (Stubbe, 1990, Adv Enzymol. Relat. Areas Mol. Biol. 63:349-419; Fontecave et al., 1992, Adv. Enzymol Relat. Areas Mol. Biol. 147-183; Sjoberg, 1995, Nucleic Acids and Molecular Biology. 9:192-224).
Necessary for RR activity, the association of the R1 and R2 subunits is completely governed by the binding of the C-terminal residues of R2 to R1, as demonstrated for E. coli RR (eRR, Climent et al., 1991, Biochemistry. 30:5164-5171; Climent et al., 1992, Biochemistry 31:4801-4807), HSV-RR (Filatov et al., 1992, J. Biol. Chem. 267:15816-15822) and mouse RR (mRR, Hamann, 1994, Purification, Characterization and Activity of Chimeric E. coli/Mouse and Plasmodium faciparum Small Subunits of Type I Ribonucleotide Reductase. Ph.D. Thesis in Chemistry, University of Pennsylvania). In addition, NMR studies of mR2 establish that the highly flexible C-terminal residues become rigid in the presence of added R1 protein (Lyckskell et al., 1994, Biochemistry. 33:2838-2842). The association constant of R1.sub.2 to R2.sub.2 is modest (.about.0.4-1.times.10.sup.7 M.sup.-1) (Climent et al., 1991, Biochemistry. 30:5164-5171; Hamann, 1994, Purification, Characterization and Activity of Chimeric E. coli/Mouse and Plasmodium faciparum Small Subunits of Type I Ribonucleotide Reductase. Ph.D. Thesis in Chemistry, University of Pennsylvania; Rova et al., 1995, Biochemistry. 34:4267-4275), and the C-terminal peptide of R2 is able to inhibit enzymatic activity by competing with R2 for association with R1 (Climent et al., 1991, Biochemistry. 30:5164-5171; Gaudreau et al., 1992, J. Med. Chem. 35:346-350; Fisher et al., 1993, J. Med. Chem. 36:3859-3862). The substantial difference between the HSV R2 C-terminal sequence (YAGAVVNDL; SEQ ID NO: 1) and the corresponding mammalian sequence (NSFTLDADF; SEQ ID NO: 2) has been exploited in the development of selective peptide inhibitors of viral RR (Liuzzi et al., 1994, Nature. 372:695-698; Moss et al., 1996, J. Med. Chem. 39:2178-2187). The feasibility of developing specific inhibitors for yeast RR (Fisher et al., 1993, J. Med. Chem. 36:3859-3862), P. falciparum RR (Rubin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:9280-9284) and eRR (Cosentino et al., 1990, Biochem. Cell Biol. 69:79-83) has also been demonstrated.
Refined crystal structures of both the R1 and R2 subunits from E. coli have been reported, at 2.7 .ANG. and 2.2 .ANG. resolution, respectively (Uhlin et al., 1993, Nature 370:533-539; Nordlund et al., 1993, J. Mol. Biol. 232:123-164). The structure of the R2 subunit reveals that the tyrosine radical, Y122, is buried inside the protein, 5 .ANG. from the nearest Fe atom. The ligands directly coordinated to the two Fe (III) atoms, namely, two histidine residues, two glutamic acid residues, and an aspartic acid residue, are highly conserved evolutionarily.
The thirty carboxy-terminal residues of the R2 subunit cannot be located in the crystal structure of E. coli R2. The structure of the R1 subunit was determined in the presence of a synthetic twenty residue peptide homologous to the twenty carboxy terminal amino acid residues of E. coli R2, although only the eight carboxy-terminal residues of R2 have been located within the structure of R1. This octamer is bound between two .alpha.-helices of R1 corresponding to residues 340-350 and 710-726 of R1.
This result is consistent with evidence that a region near the carboxy-terminus of R1 forms at least part of the conserved site for binding by R1 of the carboxy-terminal portion of R2. An azidophenyl derivative of FTLDADF (SEQ ID NO: 3) was found to photoincorporate into a peptide of mouse R1 (Davis et al., 1994, J. Biol. Chem. 269:23171-23176) corresponding to residues 724-735 of mouse R1, the sequence of which corresponds to residues 718-728 of E. coli R1. These data suggest that this region of R1 is involved in binding the carboxy-terminal portion of R2.
R1 variants, denoted A1091S and P1090L, have been isolated from strains of HSV-1 which exhibit weak resistance to an R2 carboxy-terminal peptidomimetic inhibitor (Liuzzi et al., 1994, Nature 372:695-698; Bonneau et al., 1996, J. Virol. 70:787-793). The affinities of the A1091S R1 variant for R2 and for the inhibitory peptidomimetic are decreased by factors of 10 and 25, respectively, relative to wild type HSV-1 strains. Residues 1090 and 1091 of HSV-1 R1 correspond to residues 710 and 711 of E coli R1. These results are summarized in the alignment presented below. It is noteworthy that the region of R1 implicated in binding of R2 peptide is adjacent to the most conserved sequence (presented in bold type) within R1.
______________________________________ mouse R1.sup.a PNYGKLTSMHFYGWKOGLKTGMYY re- 715-738 (SEQ ID NO: 4) si- dues HSV1- R1.sup.b IPASTLVRLLVHAYKRGLKTGMYY 1089-1112 (SEQ ID NO: 5) E. coli-R1 VPMQQLLKDLLTAYKFGVKT.LYY 709-731 (SEQ ID NO: 6) ______________________________________ .sup.a underlined residues correspond to affinity labeled peptide. .sup.b underlined residues, when mutated, confer weak resistance to nonpeptide peptidomimetic inhibitors
There is a need for the development of effective and selective ribonucleotide reductase inhibitors. Given the fact that ribonucleotide reductase enzymes are ubiquitous throughout nature, in certain instances, the use of a general inhibitor of these enzymes may have a deleterious effect in an organism to which the inhibitor is administered. It is also necessary therefore, to have the capability of generating inhibitors of specific ribonucleotide reductase enzymes, wherein inhibition of any one ribonucleotide reductase may be accomplished without significantly inhibiting other ribonucleotide reductases. Thus, there is a long felt need for the development of effective and selective inhibitors of ribonucleotide reductase activity and, in particular, for inhibitors which are designed to selectively inhibit particular ribonucleotide reductase enzymes while having a lesser or no effect on other ribonucleotide reductase enzymes. The present invention satisfies these needs.